Diagnostic method for disorders using copeptin

ABSTRACT

Disclosed herein is the use of copeptin as a diagnostic marker for the determination of the release of vasopressin, especially in connection with disorders associated with non-physiological alterations of vasopressin release from the neurohypophysis, especially for detection and early detection, diagnosing and monitoring of the course of cardiovascular diseases, renal and pulmonary diseases as well as shock, including septic shock, sepsis and diseases/disorders of the central nervous system and neurodegenerative diseases.

The present invention relates to the use of copeptin and/or itsprecursors or fragments and/or its splice variants, fragments comprisingcopeptin and/or combinations and/or derivatives thereof in medicaldiagnosis as humoral bio-marker for disorders associated with or causedby non-physiological alterations of vasopressin release from theneurohypophysis, as there are, for example, cardiac diseases, renaldisaeases, inflammatory diseases and sepsis and diseases/disorders ofthe central nervous system and neurodegenerative diseases and others asmentioned below.

In the following text all biomolecules and fragments, variants andcombinations thereof as mentioned above, which share the common featurethat they display copeptin immunoreactivity, are referred to as“copeptin” in the present application. The term “copeptin”, therefore,inter alia also comprises for example VP-prohormone, if present in asample as such.

Copeptin according to the present invention is used as biomarker,especially humoral biomarker, which can be used to diagnose disordersassociated with or caused by non-physiological alterations, espciallyincreases, of vasopressin release from the neurohypophysis as there arecardiovascular diseases like chronic or congestive heart failure,cardiac arrest, cardiac shock, cardiac infarction, acute myocardialinfarction, arterial hypertension, cardiac surgery, cirrhosis, pulmonarydisorders, kidney (renal) diseases as polycystic kidney disease,Diabetes insipidus, forms of hyponatremia, forms of syndrome ofinappropriate antidiuretic hormone secretion, hemorrhage, edema-formingstates, inflammatory diseases, trauma, burns, infectious complicationsthereof and sepsis, severe sepsis and septic shock, as well asdiseases/disorders of the central nervous system (CNS) andneurodegenerative diseases.

If, in the present application, a use as biomarker is mentioned, thismeans the determination of said biomarker in in vitro samples ofbiological fluids (i.e. ex vivo) as blood, serum or plasma and liquorcerebrospinalis (cerebrospinal fluid; CSF). For any skilled person thisclearly implies that only such physiologically occurring “copeptin”molecules are to be determined which in fact can be present in suchsamples. There may be present in a sample of a body fluid severaldistinct species of essentially identical immunoreactivity, which differe.g. in length and/or by the presence and/or type and/or degree of theirposttranslational modification, e.g. glycosylation and/orphosphorylation. In view of the inherent possibility that any givenassay may recognize more than just one sort of molecule, according to apreferred embodiment the determination of copeptin is to be understoodas determination of copeptin immunoreactivity, especially preferred asimmunoreactivity as measured with an assay as described below.

As far as the use of the present invention also extends to thepreparation of assay components and reagents useful in connection withthe determination of copeptin as biomarker, or as active ingredient inpharmaceuticals, any suitable copeptin peptides or derivatives,including fusion products and modifications having e.g. a reducedhomology with the naturally occurring copeptin, or having a modifiedstability, can be used, without any restriction to naturally occurringcopeptin products.

The term copeptin of the present invention consequently comprises alsoamino acid sequences showing e.g. only 75% homology, preferred at least80% homology, more preferred at least 90% homology to copeptin.

Terms as “diagnosis” or “diagnostic” are used in this specification asgeneral terms, which, if not defined otherwise, are intended to comprisenot only diagnosis in the sense of identifying a specific disease, butalso screening of asymptomatic or high risk populations at risk ofcertain diseases or suspected to have certain diseases, especially forearly detection, monitoring of untreated or treated patients andmonitoring the course of a therapy and for prognosis/—early prognosisand survival prognosis.

The invention further relates to antibodies raised against copeptin oragainst partial peptides of copeptin, especially for use in a method asmentioned above, as well as kits and assays involving such components.

Vasodilatory states of shock are life threatening situations. Theperipheral blood pressure decreases drastically and often does notnormalise after administration of catecholamines. The most frequent formof shock is septic shock, which is also the most severe form of sepsis.Furthermore vasodilatory shock can manifest itself after severe heartsurgery, hemorrhagic and cardiac shock or after poisoning by medicamentsor toxins [1, 2].

A series of peptides being predominantly effective via theautocrine/paracrine route are involved in the regulation of bloodpressure. The following molecules are known to have vasodilatoryfunction: e.g. adrenomedullin, calcitonin gene-related peptide (CGRP)and atrial natriuretic peptide (ANP). Vasoconstrictive effects show forexample the following molecules: endothelin, angiotensin II andvasopressin (also known as arginine-vasopressin, antidiuretic hormone(ADH)).

Vasopressin is a cleavage product of a larger precursor molecule (“VPpro-hormone”; its polypeptide sequence is shown in FIG. 3; or as SEQ IDNO:4) that is mainly formed in the neurons of the hypothalamus [20].After synthesis the VP pro-hormone is glycosylated in the Golgiapparatus, packed into secretory vesicles and split by pro-hormoneconvertase SP3 into the three peptides vasopressin, neurophysin andcopeptin. After axonal migration to the nerve endings of the hypophysisthe peptides are secreted from the vesicles upon certain stimuli (e.g.high osmolarity, decrease of blood volume or different hormones).

The most prominent physiological effect of vasopressin is the retentionof body water (antidiuresis). The effect of vasopressin tophysiologically increase blood pressure is in healthy individuals lessprominent than in septic shock. Further physiological functions ofvasopressin are the regulation of the pituitary adrenal axis (ACTH,Cortisol), stimulation of the activity of the gastro-intestinal tractand the aggregation of blood platelets [1; numbers in brakkets refer tothe attached list of literature references].

In the pathogenesis of shock vasopressin plays a central role: inexperimental shock-models it was shown that plasma concentrations ofvasopressin are increased by three orders of magnitude above the normalconcentration within 15 minutes after stimulation [4]. After rapidrelease of vasopressin stored in the hypophysis the vasopressinconcentrations are decreasing drastically during further course of shocksyndrome as was observed in patients with septic shock [5-7]. Thisobservation was the base for the concept of a vasopressin substitutiontherapy for the treatment of septic shock that was successfully tested[8-10]. These results indicate that an endogenous decrease ofvasopressin is contributing to the state of septic shock [5].

Vasopressin has also been discussed as a marker for the prognosis ofprobability of survival of patients with cardiac arrest [11] andconsequently it was used for the treatment of such patients [12].

A pathophysiological overexpression of vasopressin or VP pro-hormone hasbeen shown for several types of cancer like prostate, testicular cancer,ovarian and pancreatic cancer, pituitary adenomas and gangliomas,olfactory neuroblastomas, breast and colon tumours, nasopharyngealcarcinoma, head and neck cancer, phaeochromocytoma and tumours ofgastrointestinal origin, squamous-cell carcinomas, adenocarcinomas andlarge cell carcinomas [13, 24, 25, 26]. Vasopressin produced or releasedin cancers is to be considered as pathophysiologically formed, i.e.formed by unnormal physiological routes (altered pathological tissues)which are distinct from the normal physiological vasopressin production.

In diseases as mentioned above vasopressin is released from an organ(neurohypophysis) which is its normal origin, although innon-physiological levels.

Copeptin—also known as C-terminal glycoprotein—comprises 39 amino acids,its sugar moiety and has a molecular weight of about 2000 Da [15-17].The glycosylation is at position_(.) 131 of the precursor VP-prohormone(cf. SEQ ID NO:4). The biological function of copeptin remains unclear.

The direct determination of vasopressin as humoral diagnostic marker inbody fluids as e.g. serum or plasma itself is not suitable for routinediagnostics. More than 90% of vasopressin are bound to blood plateletsand are thus not available for the measurements [21]. Thus freevasopressin found in the plasma does not reflect the true amount ofvasopressin released into the blood stream. The binding of vasopressinto the blood platelets leads to different results, depending on theamount of platelets included in the measurement, which is variabledepending on the centrifugation used to obtain the plasma [22]. Afurther hindrance is the fact that a higher amount of vasopressin isobserved, if the blood sample is left at room temperature beforecentrifugation. These effects and the short half-life of vasopressin invivo (24 minutes, [14]) and in plasma samples ex vivo even when storedat −20 [23] so far has hindered the use of vasopressin in routinediagnostics. Due to the short half-life, it is not possible in routinediagnostics to take samples, obtain the plasma, transport the sampleinto the laboratory and do the diagnostics in the laboratory includingthe required tests before vasopressin reaches a critical level ofdetection.

Furthermore due to the low in vivo stability of vasopressin and thevariable results due to the binding to and release from blood platelets,the use as a biomarker is extremely limited even under optimized sampleslogistics, as the influence of the degradation occurs rapidly.

The object of the invention was to overcome the effects of thedisadvantageous half-life of vasopressin and the variable results inmeasurements and to develop a method, use and a kit for the detectionand determination of the molecules associated with the release ofvasopressin, more specially copeptin, for the diagnosis ofcardiovascular diseases and sepsis.

This object could be achieved on the basis of the surprising findingthat certain fragments of VP prohormone—the precursor ofvasopressin—copeptin in particular, can be used as a tool for thedetermination of the physiological release of vasopressin, in particularin body fluids, in the diagnosis and monitoring of cardiovascualrdiseases and sepsis.

This generation of the fragments, copeptin in particular, correlateswith the release of vasopressin, since all are derived from the sameprecursor.

Furthermore the stability of the precursor proteins, fragments and/orcombinations thereof ex vivo were found to be surprisingly high andrender the fragments, copeptin in particular, fully suitable for routinepurposes.

This linkage between the fragments like copeptin and other fragments ofthe precursor peptides made them suitable as diagnostic tools for alldiseases, where vasopressin plays a role. Copeptin in particular cantherefore be used in mediacal diagnostics for diagnosing and monitoringa variety of diseases, especially cardiovascular diseases and systemicinflammations, especially sepsis.

Furthermore the present invention in one embodiment relates to the useof copeptin for diagnosis of the diseases, course control and prognosisfor the above mentioned diseases where vasopressin plays a role.

Clinical data may additionally be taken into consideration to supportthe determination of the disease.

It is possible to use amino acid sequences showing at least 75%homology, preferred 80% homology more preferred at least 90% homology tocopeptin according to the present invention.

In a preferred embodiment of the present invention according to thefollowing examples two regions (PATV17; PLAY17) of the humanvasopressin-neurophysin 2-copeptin-precursor amino acid sequence werechosen as immunological binding sites to be recognized by specificantibodies:

positions 132-147: (PATV17-SEQ ID NO: 1) CATQLDGPAGALLLRLVpositions 149-164: (PLAY17-SEQ ID NO: 2) CLAGAPEPFEPAQPDAY,

For calibration purposes and for the preparation of standard solutions,a peptid comprising both binding sites mentioned above was used (PAY33):

positions 132-164: (PAY33-SEQ ID NO: 3)ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY.

The immunological binding sites PATV17 and PLAY17 to be recognized byspecific antibodies were selected such that they do not include theputative glycosylation site of copeptin at position 131. Therefore, thepresence or absence of a posttranslational modification (glycosylation)of the determined copeptin should not have any significant impact on therecognition of copeptin in an assay using such antibodies. The term“copeptin”, therefore, includes the “naked” copeptin peptide as well asposttranslationally modified forms of said peptide.

When each of the pepides mentioned above were synthesized as describedbelow, an amino terminal cystein residue was added to each copeptinamino acid sequence, and the peptides were chemically synthesized assoluble proteins according to methods known by the person skilled in theart. They were purified and quality controlled by mass spectroscopy andreversed phase HPLC and lyophilised into aliquots (Jerini AG, Berlin,Germany).

The synthesized peptides according to the present invention were used toproduce antigens and injected into animals to raise antibodies againstcopeptin according to the present invention. Different methods can beused to achieve this object known by the person skilled in the art.

In a preferred embodiment the peptides PATV17 and PLAY17 were conjugatedto a carrier protein keyhole limpet hemocyanin (KLH) via MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester) according to the methodsof Pierce, Rockford, Ill., USA. Antibodies were produced to the abovementioned peptides in sheep. In a preferred embodiment of the presentinvention, polyclonal antibodies were raised against the above mentionedpeptides. Antibodies were purified according to known methods. In apreferred embodiment of the invention, this was achieved preferably byligand specific affinity chromatography by coupling the peptides via theamino terminal cystein residue to SulfoLink-Gel of Pierce (Boston, USA)according to the methods of Pierce. In a preferred embodiment theantibodies were tagged with a marker to enable detection. The markerused is preferably a luminescent marker and in a yet more preferredembodiment, the antibody was tagged with a chemiluminescent marker andin a yet further preferred embodiment the antibodies against PATV17(0413-pAK) and PLAY17 (0417-pAK) were tagged with a chemiluminescentmarker.

The invention in a further preferred embodiment involves the use of thegenerated antibodies for detection of copeptin in accordance with thepresent invention in samples of body fluids, as well as a kit comprisinga certain quantity of such an antibody or more antibodies specific todetect molecules in accordance with the present invention. Differentassays can be used to detect the molecules as are known to the personskilled in the art comprising competitive or sandwich immunoassays inmanual, automated or point of care test formats employing various kindsof labels.

Methods for the detection of binding of the antibody to the respectivemolecule are also known by the person skilled in the art. All such knownassay formats can be used in the context of the determination ofcopeptin. It is, for example, within the scope of the present inventionto determine copeptin with the aid of a rapid test device, e.g. of theimmunochromatographic type, as so-called POC (Point of Care) test. Thedetermination of copeptin can also be conducted with a homogeneous assayof the so-called KRYPTOR® type, using the so-called TRACE® technology.

A preferred embodiment of the present invention discloses the use ofantibodies generated against the above mentioned peptides, 0413-pAK and0417-pAK, in particular for a two-site immunoassay of the sandwich type.Another preferred embodiment of the invention discloses the use of theseantibodies for the detection and the determination of the concentrationof the molecules of the present invention, copeptin in particular invarious body fluids and other biomaterials. In a preferred embodimentcopeptin can be detected at concentrations above 50 pg/ml of body fluid(FIG. 1).

In one embodiment the invention is based on and uses the discovered longterm stability of copeptin ex vivo in plasma and serum (Table 2). Inplasma and serum copeptin levels were surprisingly stable even after twodays storage at room temperature. Thus copeptin is by far more suitablefor diagnostic purposes than vasopressin.

A preferred embodiment of the invention discloses the use of antibodiesgenerated against PLAY17 and PATV17 for the detection of copeptin inhealthy individuals, in patients with sepsis, cardiac infarction andincreased arterial blood pressure (FIG. 2), and for determining theseverity of chronic or congestive heart failure (CHF) (FIG. 4).

The invention further permits the determination of the presence andstability of the molecules of the present invention, copeptin inparticular in body fluids, and the determination of the difference inpeptide concentration in healthy controls and patients of variousdiseases comprising those mentioned above (FIG. 2; FIG. 4). The medianof healthy control individuals is at about 13 pg/ml.

The invention further discloses a significant change of theconcentration of the humoral biomarker copeptin in body fluids in stateof disease comprising those mentioned above.

A preferred embodiment of the invention is based on the surprisingfinding of a highly significant change i.e. an about 10 fold increase incopeptin concentration in plasma of sepsis patients (median 150,5 pg/ml)and in cardiac infarction (median 129,5 pg/ml) and an about 35 foldincrease in patients with increased arterial blood pressure (median459,5 pg/ml).

In patients with CHF the measured levels of copeptin correlates wellwith the severity of the illness which is generally evaluated using theNew York Heart Association (NYHA) functional classification system,wherein NYHA classes I to IV correspond to the following typicalfunctional capacities: NYHA class I—asymptomatic; NYHA class II—symptomswith moderate exertion; NYHA class III—symptoms with minimal exertion;NYHA class IV—symptoms (dyspnea) at rest. In a study in which copeptinlevels in plasma samples of a total of 348 CHF patients (25 in NYHAclass I; 124 in NYHA class II; 127 in NYHA class III; 72 in NYHA classIV; see Table 1) were determined, the medians for the different classesshowed a clear tendency (see Table 1 below).

The invention also provides a diagnostic method, kit and assay for theabove mentioned diseases, using one or more antibodies of copeptin inparticular.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the standard curve for the sandwich-immunoassay forcopeptin immunoreactivity using the peptide PAY33 as a standardmaterial.

FIG. 2 shows the concentration of copeptin immunoreactivity in samplesof healthy individuals and different groups of patients (sepsis, cardiacinfarction and increased arterial blood pressure).

FIG. 3 shows the vasopressin prohormone amino acid sequence (one-lettercode; cf. also SEQ ID NO:4).

FIG. 4 shows the concentrations of copeptin immunoreactivity in samplesof patients with CHF (chronic heart failure) classified according to theNYHA classification system.

MATERIALS, METHODS AND MEASUREMENTS EXAMPLE 1

Peptide Synthesis

Peptides were synthesized and their quality was controlled by massspectrometry and reversed phase HPLC and lyophiliysed in aliquots(Jerini AG, Berlin, Germany) according to standard procedures known tothe person skilled in the art. The amino acid sequences of the peptidesare the following (numbers refer to corresponding positions in the humanprovasopressin-neurophysin 2-copeptin-precursors (positions 132-147 and149-164):

PATV 17 (132-147 + N-terminal cystein residue): [Sequence ID 1]CATQLDGPAGALLLRLV, PLAY 17 (149-164 + N-terminal cystein residue):[Sequence ID 2] CLAGAPEPFEPAQPDAY, Standard peptide PAY 33 (132-164)[Sequence ID 3] ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY.

EXAMPLE 2

Conjugation and Immunization

Peptides of Sequence IDs 1-2 were conjugated to the carrier protein KLH(keyhole limpet hemocyanin) by MBS(-Maleimid-obenzoyl-N-hydroxysuccinimid ester) according to theprotocols for “NHS-esters-maleimide crosslinkers” by PIERCE, Rockford,Ill., USA. Sheep were immunized receiving 100 μg of conjugate (μgaccording to the peptide content of the conjugate) and subsequently 50μg of conjugate every four weeks (quantitiy related to the peptidecontent of the conjugate). Starting at month 4 after immunization everyfour weeks 700 ml of blood were withdrawn from every sheep and antiserumwas gained by centrifugation. Conjugation, immunizations and productionof antisera were done by MicroPharm, Carmerthenshire, UK.

EXAMPLE 3

Purification of Antibodies

The polyclonal antibodies from sheep were purified using ligand specificaffinity purification. For that step the peptides PATV 17 and PLAY 17were linked to SulfoLink-Gel supplied by Pierce (Boston, USA). Thebinding occurred according to the protocol of Pierce. 5 mg of peptidewere added per 5 ml of gel.

In summary, columns were washed three times with 10 ml elution buffer(50 mM citric acid, pH 2.2) and binding buffer (100 mM sodium phosphate,0.1% Tween, pH 6.8). 100 ml of sheep antiserum were filtered using afilter diameter of 0.2 μm and added to the column material, which hadbeen transferred from the column to a beaker with 10 ml binding buffer.The material was incubated over night at room temperature by gentlerotation. The material was transferred to empty columns (NAP 25,Pharmacia, emptied). The eluates were discarded. Subsequently thecolumns were washed with 250 ml protein-free binding buffer (proteincontent of washed eluate <0.02 A 280 nm): Elution buffer was added tothe washed columns and fractions of 1 ml were collected. The proteincontent of each fraction was determined by the BCA-method (according tothe protocol of PIERCE, Rockford, Ill., USA). Fractions of a proteincontent >0.8 mg/ml were pooled. After determination of protein content39 mg of anti-PATV 17 antibody 0413-pAk and 103 mg of anti-PLAY 17antibody 0417-pAk were gained.

EXAMPLE 4

Tagging

The anti-PLAY 17 antibody 0417-pAk was treated as follows: 500 μl ofaffinity purified antibodies generated were rebuffered in 1 ml 100 mMpotassium phosphate buffer (pH 8.0) via a NAP-5 gel filtration column(Pharmacia) according to the protocol of Pharmacia. The proteinconcentration of antibody solution was 1,5 mg/ml.

For the tagging with a chemiluminescent marker 10 μl ofMA70-Akridinium-NHS-ester (lmg/ml; Hoechst Behring) were added to 67 μlof antibody solution and incubated for 15 minutes at room temperature.Then 423 μl of 1 M glycin was added and incubated for 10 minutes. Thesolution was rebuffered in 1 ml solvent A (50 mM potassium phosphate,100 mM NaCl, pH 7.4) using a NAP-5 gel filtration column according tothe protocols of Pharmacia. For final elimination of unbound label a gelfiltration HPLC was done (Column: Waters Protein Pak SW300). The samplewas added and chromatographed at a flow rate of 1 ml/minute in solventA. The flow was continuously monitored in a UV-meter at wave length of280 and 368 nm to determine the degree of tagging. The absorption ratio368/280 nm of labelled antibody was 0,1. The fractions containingmonomeric antibodies were collected (retention time 8-10 minutes) andtaken up in 3 ml 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serumalbumin, 0,1% sodium azide, pH 7.4)

EXAMPLE 5

Coupling

The anti-PATV 17 antibody 0413-pAk was immobilized on irradiated 5 mlpolystyrol tubes (Greiner, Germany). For that procedure the antibodysolution was diluted to a protein concentration of 6.6 μg/ml with 50 mMTris, 100 mM NaCl, pH 7.8. 300 μl of diluted protein solution per tubewere pipetted. These were incubated for 20 hours at 22° C., the solutionwas removed. Then 4.2 ml of a 10 mM sodium phosphate, 2% Karion FP, 0.3%bovine serum albumin, pH 6.5 solution were added to each tube. After 20hours the solution was removed and the tubes were dried in a vacuumdrier.

EXAMPLE 6

Immunoassay

The following assay buffer was used: 100 mM sodium phosphate, 150 mMNaCl, 5% bovine serum albumin, 0.1% unspecified sheep IgG, 0.1% sodiumazide, pH 7.4.

The copeptin concentration of EDTA-plasma of healthy individuals andpatients of various diseases/diseases mentioned above was determined,heart diseases and diseases of the circulation in particular.

As a standard material chemically synthesized peptide (peptide PAY 33)was used which corresponds to positions 132-164 ofvasopressin-neurophysin 2-copeptin precursor. The standard was dilutedin normal horse serum (Sigma).

In the test tubes 100 μl of standards or sample as well as 100 μl ofassay buffer was pipetted. The tubes were incubated for two hours at 22°C. using gentle rotation. After washing 4 times with 1 ml of washingbuffer (0.1% Tween 20), the supernatant was discarded. Then 200 μl ofassay buffer, containing 1 million RLU (relative light units) ofMA70-tagged antibody was added and incubated for a further two hoursunder gentle rotation at 22° C. After washing 4 times with 1 ml ofwashing buffer (0.1% Tween 20), the chemiluminescence bound to the tubewas determined in a luminometer (Berthold, LB952T, basic reagents BrahmsAG). Using the software MultiCalc (Spline Fit) the concentrations of thesamples were determined.

EXAMPLE 7

Determination of Copeptin Concentration

The term copeptin immunoreactivity describes the amount of substratedetected by the developed sandwich immunoassay. The sandwich immunoassayuses antibodies raised against positions 132-147 and 149-164 of thevasopressin-neurophysin 2-copeptin-precursor for detection of thesubstrate. A typical standard curve for the developed assay is describedin FIG. 1. Using the assay concentrations above 50 pg/ml copeptinimmunoreactivity in plasma or serum can be determined quantitatively.

EXAMPLE 8

Concentration of Copeptin Immunoreactivity in Healthy Individuals andState of Disease

Serum and plasma of healthy individuals and patients suffering fromvarious diseases comprising sepsis, cardiac infarction and increasedarterial blood pressure were analysed (FIG. 2): The copeptinimmunoreactivity was determined. Compared to healthy individualscopeptin immunoreactivity was surprisingly increased in state ofdisease. Healthy individuals showed a median of about 13 pg/ml, sepsispatients a median of about 150 pg/ml, in cardiac infarction the medianwas about 129 pg/ml and in increased arterial blood pressure the medianwas about 459 pg/ml.

EXAMPLE 9

Concentration of Copeptin Immunoreactivity in Patients with ChronicHeart Failure (CHF) of NYHA Classes I to IV

In serum and plasma samples of a total of 348 CHF patients (25 in NYHAclass I; 124 in NYHA class II; 127 in NYHA class III; 72 in NYHA classIV; see Table 1) copeptin levels were determined using the assaydescribed above. The results are shown in diagrammatic form in FIG. 4.As can be seen, the medians of the copeptin concentration in pg/ml forthe different classes showed a clear tendency to increase in that themedian for patients of NYHA class I was found to be 20.30 pg/ml, classII 33.25 pg/ml, class III 49,60 pg/ml, and class IV 85.80 pg/ml (see thestatistical data in Table 1 below).

TABLE 1 NYHA I NYHA II NYHA III NYHA IV Number of 25 124 127 72 valuesMedian 20.30 33.25 49.60 85.80 Mean 27.00 45.32 63.91 184.7 Lower 95% CI17.45 38.79 55.29 118.9 of mean Upper 95% CI 36.56 51.86 72.54 250.5 ofmean CI = confidence interval

The finding that there is a close correlation of the severity of CHFwith the copeptin levels in plasma makes copeptin a biomarker candidatefor use in the diagnosis (positive or negative diagnosis) of CHF, themonitoring of the course and evolution of CHF and the monitoring andcontrol of a CHF therapy. Further, in view of current attempts toevaluate the usefulness of vasoporessin receptor antagonists in thetherapy of heart failure [27], the determination of copeptin in serumor. plasma samples of heart failure patients can allow theidentification of such patients who would benefit more than others froma treatment with vasopressin receptor antagonists.

EXAMPLE 10

Stability of Copeptin Immunoreactivity

Copeptin immunoreactivity was found to be surprisingly stable in plasmaand serum (Table 2). Table 2 shows the ex vivo stability of endogenousimmunoreactive copeptin in serum and plasma of sepsis patients.

TABLE 2 Storage Recovery Sample (days/temperature) (%) Serum 1 d/4° C.98.0% (n = 3) 2 d/4° C. 99.2% 1 d/RT 94.1% 2 d/RT 103.7% Plasma 1 d/4°C. 103.4% (n = 5) 2 d/4° C. 101.6% 1 d/RT 99.9% 2 d/RT 104.9%

Even after two days storage at room temperature (RT) no decrease inimmunoreactivity could be detected.

Thus the ex vivo stability of copeptin immunoreactivity is surprisinglyremarkably increased as compared to vasopressin.

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1. A diagnostic in vitro method for the diagnosis and monitoring ofdisorders associated with or caused by non-physiological alterations ofvasopressin release from the neurohypophysis by determining in a sampleof a body fluid of the patient the amount of copeptin and/or apathophysiologically occurring precursor, splice variant, fragment orposttranslationally modified form of copeptin displaying copeptinimmunoreactivity, and associating the determined amount of copeptin orcopeptin immunoreactivity with the presence and/or course and/orseverity and/or prognosis of such disorder.
 2. The method of claim 1,wherein said disorder associated with or caused by non-physiologicalalterations of vasopressin release from the neurohypophysis is selectedfrom the group consisting of chronic or congestive heart failure,cardiac arrest, cardiac shock, cardiac infarction, acute myocardialinfarction, arterial hypertension, cardiac surgery; cirrhosis; pulmonarydisorders; kidney (renal) diseases as polycystic kidney disease;Diabetes insipidus; forms of hyponatremia, forms of syndrome ofinappropriate antidiuretic hormone secretion; hemorrhage, edema-formingstates, inflammatory diseases, trauma, burns, infectious complicationsthereof and sepsis, severe sepsis and septic shock; anddiseases/disorders of the central nervous system (CNS).
 3. The method ofclaim 1, wherein the copeptin immunoreactivity is an immunoreactivitywhich can be determined by using a sandwich immunoassay using a firstspecific binder recognizing an amino acid sequence present in SEQ IDNO:1, and a second specific binder recognizing an amino acid sequencepresent in SEQ ID NO:2.
 4. The method of claim 3, wherein said specificbinders are polyclonal and/or monoclonal antibodies.
 5. The method ofclaim 3, wherein said immunoassay is a heterogenous immunoassay using afirst immobilized specific binder and a second solubilized specificbinder which carries a detectable label or which can be selectivelylabeled by reaction with a labeled marker molecule.
 6. The method ofclaim 3, wherein said immunoassay is a homogeneous immunoassay.
 7. Themethod of claims 1, wherein the biological fluid is selected from serum,plasma, blood or (cerebrospinal fluid; CSF).
 8. The method of claim 2,wherein said disorder is selected from the group consisting of sepsis,cardiac infarction, chronic heart failure and increased arterial bloodpressure. 9-12. (canceled)